Electroendosmosis, also referred to as electroendosmotic or electroosmotic flow, is a phenomenon which frequently occurs in electrophoretic separations of solute ions dissolved in a solvent or solvent system. Electroendosmosis is particularly pronounced in electrophoresis which is performed in capillaries made of a silica-containing material. Electroendosmosis causes bulk flow of the solvent system in response to the electric field, independently of the electrophoretic migration of the solute ions themselves which occurs at rates varying with the charge-to-mass ratio and the polarity of each ion. The bulk flow impairs the separation of solutes since it causes mobilization of all solutes at a common rate as part of the solution in which they are dissolved, thereby adding a nondifferentiating component to their mobility. This effectively shortens the path of travel attributable to electrophoresis itself, thereby lessening the degree of eleetrophoretic separation for a column of given length. In extreme cases, electroendosmosis causes peak broadening and loss of resolution. Electroendosmosis also impairs the reproducibility of a separation when repeated runs are performed in the same cell, column or capillary, since small amounts of solutes retained after the separation is terminated tend to alter the electroendosmotic effect, and the degree to which solutes retained from one separation affect subsequent separations depends on the balance between retention of the solutes within, and their release from, the separation region.
Until now, it has been believed that electroendosmosis arises solely from an electrokinetic potential existing between a solid surface such as the surface of a capillary wall or the surface of a bead in a packed bed and the liquid phase adjacent to the solid surface. As a result, electroendosmosis in capillaries is commonly suppressed by a coating on the interior capillary surface. The coating generally consists of neutral or charged groups covalently bound to the capillary surface, eliminating charged groups which were otherwise exposed on the surface and shielding the liquid medium adjacent to the wall from charged groups located near the surface which are not directly bonded to the coating material. Coatings are not an ideal means of eliminating electroendosmosis, however, since electroendosmosis develops in coated capillaries as well after repeated use. This is presumed to be attributable to a deterioration of the coating upon extended use or upon exposure to harsh solutes or separation media, or to the adsorption of charged analytes from previous experiments. The deterioration limits the useful life of a coated capillary. When capillaries with partially deteriorated coatings are used in isoelectric focusing, for example, the deterioration limits the length of the focusing time for any single run.
Another method which has been used to reduce electroendosmosis in capillaries is the inclusion of a small quantity of a cellulose derivative in the separation medium to raise the viscosity of the medium. This unfortunately affects the rate of migration of the solutes as well, and merely retards rather than eliminates the electroendosmotic effect.
A still further method is the application of highly charged hydrophobic polymers to coat the wall of the capillary prior to application of the samples, as disclosed by Wiktorowicz, U.S. Pat. No. 5,181,999, issued Jan. 26, 1993. Polymers such as Polybrene (N,N,N',N'-tetramethyl-1,6-hexanediamine polymer with 1,3-dibromopropane) have been used for this purpose. These polymers must be tightly bound to the wall, however, and the capillary must be pre-equilibrated with the separation electrolyte before the separation is performed. With protein analytes, it is important that the binding and equilibration be conducted prior to the introduction of the sample, to avoid having any residual Polybrene enter the analyte solution where uncontrollable interactions of the proteins with residual Polybrene will occur.